Use of anethole dithiolethione in lung cancer chemoprevention

ABSTRACT

The present invention relates to the use of 5-(p-methoxyphenyl)-1,2-dithiole-3-thione or of a pharmaceutical derivative thereof for the preparation of a medicament for preventing lung cancer in a mammalian subject, especially for preventing or reducing the appearance of new dysplastic lesions, or the progression of pre-existing dysplastic lesions in the subject, or for improving regression of existing dysplastic lesions.

The present invention relates to the use of anethole dithiolethione inlung cancer chemoprevention.

Lung cancer is the most common cause of cancer death worldwide with amortality rate exceeding that of colon, breast and prostate cancerscombined (Greenlee et al., 2001). Former heavy smokers retain anelevated risk for lung cancers even years after they stop smoking(Halpern et al., 1993 ; Tong et al., 1996). With a large reservoir ofcurrent and former smokers and the increasing incidence of lung cancersamong women, lung cancer will remain a major health issue for the nextseveral decades.

One potential cancer control strategy for those who are at risk ofdeveloping lung cancer is to use chemopreventive agents to inhibit thedevelopment of invasive cancer either by blocking the DNA damage thatinitiates carcinogenesis or by arresting or reversing the progression ofpremalignant cells in which such damage has already occurred (Sporn etal., 1976 ; Hong et al., 1997).

Anethole dithiolethione or 5-(p-methoxyphenyl)-1,2-dithiole-3thione(Sialor®, Sulfarlem®) and 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione(Oltipraz) belong to the dithiolethiones chemical class of organosulfurcompounds with antioxidant, chemotherapeutic, radioprotective andchemopreventive properties (Kensler et al., 1992). They represent aunique class of compounds for which anti-carcinogenic activity in-vivowas predicted from biochemical measurements which include induction ofcarcinogen detoxification enzymes. In animal carcinogenesis models, thedithiolethiones exert chemoprotective activity against development oflung as well as other cancers (Kensler et al., 1992 ; Reddy get al.,1993 ; Kensler et al., 1987 ; Bolton et al., 1993 ; Pepin et al., 1992).Oltipraz has been investigated in humans, but is not an approved drug.Recently, Oltipraz was found to be too toxic for chemoprevention(Pendyala et al., 2001). On the contrary, anethole dithiolethione (ADT)is an approved drug that has been used worldwide. In Canada, Europe andother countries, ADT is used for the treatment of drug—orradiation—induced hyposalivation, in the treatment of xerostomia fromother causes (Remick et al., 1983; Epstein et al. 1983)), and is alsomarketed as a choleretic and hepatoprotective agent. At the therapeuticdose used, 25 mg three times daily, the product is safe and effective.

The present invention is based on the results of the first Phase libstudy in humans examining the potential efficacy of anetholedithiolethione (Sialor®, Sulfarlem®) in smokers with premalignantlesions in the bronchial tree.

The present invention is directed to the use of5-(p-methoxyphenyl)-1,2-dithiole-3-thione (also called anetholedithiolethione or ADT) or a pharmaceutical derivative thereof for theprevention of lung cancer.

The medicament is more particularly intended for preventing or reducingthe appearance of new dysplastic lesions, or the progression ofpre-existing dysplastic lesions in the subject.

The medicament is advantageously also intended for improving regressionof existing dysplastic lesions.

The present invention further relates to a method for preventing lungcarcinogenesis comprising administering to a mammalian subject havingprecursors of lung cancer and preferably not having cancer, especiallylung cancer (i.e. having no history of lung cancer), a pharmaceuticalcomposition comprising a therapeutically effective amount of5-(p-methoxyphenyl)-1,2-dithiole-3-thione or a pharmaceutical derivativethereof.

The terms “pharmaceutical derivative thereof” includes pharmaceuticallyacceptable salts and metabolites of5-(p-methoxyphenyl)-1,2-dithiole-3-thione.

The terms “precursors of lung cancer” as used herein includes bronchialdysplasia, metaplasia or premalignant lesions in the bronchial tree.

“A therapeutically effective amount ” as used herein refers to thatamount which provides therapeutic effect for a given condition andadministration regimen. Such compositions are liquids or otherwise driedformulations and include suitable diluents, preservatives, solubilizers,emulsifiers, adjuvant and/or carriers.

The composition can be administered to a subject by, for example,intraveneous, intraarterial, or intramuscular injection of a liquidpreparation, oral or administration of a liquid or solid preparation.Additionally, this composition can be administered by pulmonary or nasalroute.

The 5-(p-methoxyphenyl)-1,2-dithiole-3-thione or a pharmaceuticalderivative thereof is preferably administered at a dosage of about 0.1mg/kg of subject weight/day to about 50 mg/kg of subject weight/day.Preferably, the therapeutic oral dosage is of 25 mg to 100 mg once tothree times daily for an adult male or female of average weight. Morepreferably, the therapeutic oral dosage is of 25 mg once to three timesdaily for an adult male or female of average weight.

The below FIGURE and example illustrate the invention without limitingits scope.

LEGEND OF THE FIGURE

The FIGURE shows a flow diagram of subjects progressed through phases ofthe randomized trial.

One subject in the placebo group were excluded from analysis because thefollow-up biopsy of the same site that showed dysplasia prior totreatment had an incomplete epithelium making it impossible to grade theresponse.

EXAMPLE A Phase II Clinical Trial of Anethole dithiolethione (Sialor®)in Smokers with Bronchial Dysplasia

In the present study, the inventors performed a randomized, doubleblind, placebo-controlled, Phase IIb clinical trial to determine theefficacy and safety of ADT as a chemopreventive agent in smokers withpremalignant lesion of the bronchial epithelium or dysplasia.

Methods

One hundred and one volunteer current and former smokers with ≧30pack-years smoking history and bronchial dysplasia identified byautofluorescence bronchoscopy directed biopsies were randomly assignedto receive ADT 25 mg orally TID or placebo for six months followed by arepeat bronchoscopy and biopsy of the same sites plus any new areassuspicious of dysplasia. Changes in the histopathology grade of thebiopsies were used as the primary endpoint biomarker. All P values weretwo-sided.

Clinical Trial Protocol

The flow diagram of subjects progressed through the phases of therandomized trial is shown in the annexed FIGURE.

Assessment For Eligibility. Five hundred and fifty-eight current andformer smokers over 40 years of age in the Greater Vancouver area with asmoking history of ≧30 pack-years were invited to take part in thestudy. A former smoker was defined as one who had given up smoking for ayear or more. They were recruited through the community outreach networkof the public relations department of the British Columbia Cancer Agency(BCCA) using television programs, radio broadcasts and local newspapers.Following an initial interview, which included a questionnaire todocument the smoking history, a sputum sample was obtained by inductionusing simultaneous high frequency chest wall oscillation with the ABIvest (Advanced Respiratory Inc. St. Pauls, Minn.) and 3% hypertonicsaline for 12 minutes. Three hundred and seventeen subjects were foundto have sputum atypia by computer-assisted image analysis. Of these, 262agreed to have an autofluorescence bronchoscopy with the LIFE-Lungdevice (Xillix Technologies Corp., Richmond, BC, Canada) (Lam et al.,1998; Lam et al., 1999). Biopsies were taken from areas with abnormalfluorescence (Lam et al., 1998; Lam et al., 1999). In addition, at leasttwo control biopsies were taken from an upper or lower lobe. The averagenumber of biopsies per subject was 6.7 (range 4 to 12).

One hundred and fifty-six subjects were found to have one or more sitesof bronchial dysplasia on fluorescence bronchoscopy directed bronchialbiopsies. One hundred and twelve subjects with bronchial dysplasiaagreed to take part in the clinical trial.

Randomization. Participants with one or more sites of bronchialdysplasia were randomly assigned to receive either Sialor® (SolvayPharma, France) at a dose of 25 mg three times daily by mouth or itsplacebo for six months. The placebo tablets were identical in size,shape and color to the active drug. Randomization codes were generatedby a statistician at the Population and Prevention Oncology Division ofBCCA and distributed to the BCCA pharmacy. All study personnel wereblinded to the study coeds. Randomization was stratified according tothe smoking status (current or former smoker) and dysplasia grade (mild,moderate or severe). Once a participant met the enrollment criteria, thestudy nurse contacted the pharmacist who then provided the studymedication according to the randomization code.

Follow Up. The participants were seen monthly for examination of drugrelated adverse events. Liver enzymes were measured at baseline and atmonths 3 and 6 for toxicity monitoring. Fluorescence bronchoscopy wasrepeated after 6 months of study medication. The bronchoscopist wasblinded to the intervention assignment. All previously biopsied siteswere identified and re-biopsied under fluorescence bronchoscopy. Biopsywas also taken from new areas with abnormal fluorescence.

During the study, the current smokers were encouraged to stop smoking.They were invited to take part in the Fresh Start Program at the BritishColumbia Cancer Agency.

The study was approved by the Clinical Investigations Committees of theBritish Columbia Cancer Agency and the University of British Columbia.Informed consent was obtained from all the participants.

Outcomes

The primary outcome of the study was changes in the histopathology gradeof the bronchial biopsies before and after six months of intervention.The secondary endpoint was changes in the nuclear morphometry index ofthe bronchial biopsies.

Pathology of Bronchial Biopsies

The biopsies were fixed in buffered formalin, embedded in paraffin,sectioned and stained with hematoxylin-eosin (H&E). Two pathologists(JCL, AFG) systematically reviewed them. The pathologists were blindedto the treatment assignments. All biopsies were classified into one ofseven groups. Normal: as represented by pseudostratified ciliatedcolumnar epithelium. Basal cell hyperplasia: as represented by anincrease in the number and stratification of normal-appearing basalcells still covered with normal ciliated or mucin secreting cells.Metaplasia: as represented by a stratified epithelium and cytoplasmicchanges consistent with squamoid differentiation. Dysplasia: mild,moderate or severe dysplasia and carcinoma in-situ were classifiedaccording to WHO criteria (Travis et al., 1999).

To resolve minor differences, the two pathologists consulted each otherby telephone. To resolve major differences, both pathologists reviewedthe slides again and, if necessary, reached consensus diagnosis aftercommunication verbally or in person.

Nuclear Morphometry

The method of quantitative nuclear morphometry had been described indetail previously (Lam et al., 1998; Doudkine et al., 1995 ; Garner etal., 1994). McAulay et al., 1998 also suggested that high resolutionquantitative morphometric measurements may be a good intermediateendpoint biomarker for chemoprevention studies of intraepithelialbronchial neoplasia. Morphometric measurements provide a more objectiveand reproducible means for the grading of bronchial biopsies thansubjective assessment does. The device used to make thesehigh-resolution quantitative morphometric measurements was aCyto-Savant™ system (Lam et al., 1998 ; Doudkine et al., 1995; Garner etal., 1994). The images were interactively collected by an experiencedcytotechnologist under the direction of a pathologist (JCL). The imageswere collected using a 20x objective with a numerical aperture of 0.75.Analysis of the nuclei in each tissue consisted of five steps 1)focusing the field of view, 2) automatically segmenting the nuclei inthe field, 3) interactively correcting segmentation errors, 4) selectingand classifying the cell nuclei into the categories of basal,intermediate or superficial cells depending or their location within thebiopsy where possible, and 5) automatically collecting individuallyfocused images of each selected cell. Additionally, approximately 30leukocytes were collected to normalize the images for sample to samplevariations in staining intensity. On the average, greater than 100 cellnuclei were collected in this fashion for each of the biopsies. Usingbulk nuclear features such as the total area and the total amount oflight absorbed (which corresponds to the amount of DNA), shape featuresand descriptions of the distribution of stained DNA (texture features)(Doudkine et al., 1995), a scale was defined with normal nuclei definingone end and cancer-like nuclei defining the other. From the proportionof nuclei along this scale, a Morphometry Index (MI) was derived foreach biopsy (Lam et al., 1998).

Sample Size

Information from the placebo arm of a previous trial (retinol versusplacebo, NCI U01 CA68381) in this population was used to estimate thespontaneous regression rate of bronchial dysplasia. On a subject bysubject analysis, the complete response rate in 38 subjects was 24%.Assuming an increase in this rate of regression of 30% (to 54%) in theADT arm and specifying a power 0.80 for a 2-sided test at a significancelevel of 0.05 requires a sample size of 49 subjects per arm. It was alsoanticipated that each subject would present with an average of 2.4dysplastic lesions. It was anticipated that this represented anequilibrium condition with the number of new sites being approximatelyequal to the number of regressing sites (data from the U01 study). Theinventors modeled the appearance of new sites by a Poisson processparameter λt, where t was elapsed time and λ a constant, and modeled thedisappearance of existing sites by a pure death process with transitionrates dn=×n, where n is the number of existing sites and d a constant.The resulting stochastic process, D(t), describing the number ofdysplastic sites had mean, μ(t), where:${\mu(t)} = {\frac{\lambda}{d\quad}\left\lbrack {1 - {\mathbb{e}}^{- {dt}}} \right\rbrack}$and variance equaled to the mean. Assuming the subjects were in anapproximately stable state, the inventors approximated this by D(t) ast→∞, D(∞). This distribution was well approximated by a Poissondistribution with parameter λ/d and could be used to estimate the samplesize required to measure a change in the total number of dysplasticsites between the two arms. At the 0.05 significance level (2-sided) 50subjects per arm will have power 0.97 to detect a 50% reduction in thenumber of lesions per subject (2.40 to 1.20), 0.87 to detect a 40%reduction and 0.64 to detect a 30% reduction in the number lesions.

To allow for a 10% dropout rate, it was planned that a total of 110subjects would be randomized onto the clinical trial.

Statistical Analyses

The primary end-point of the study was change in the histopathologygrade based on the risk of progression to invasive cancer fromlongitudinal studies using exfoliated sputum cells and bronchialbiopsies Frost et al., 1986; Saccomanno et al., 1982; Risse et al.,1988; Melamed et al., 1982; Thiberville et al., 1997; Shibuya et al.,2001).

For the lesion-specific analysis, complete response (CR) was defined byregression of the dysplastic lesion to hyperplasia/normal. Progressivedisease (PD) was defined as appearance of lesions that were milddysplasia or worse, irrespective of whether the site was biopsied atbaseline or worsening of the dysplastic lesions present at baseline bytwo or more grades (e.g. mild dysplasia to severe dysplasia or worse).Partial response (PR) and stable disease (SD) referred to sites thatwere not CR or PD. refers to sites that are not a CR or PD. Thesestringent criteria to define regression or progression were based on ourquantitative microscopy study that showed a significant overlap in theclassification of metaplasia and mild dysplasia as well as betweenmoderate and severe dysplasia using conventional histopathology (Lam etal., 1998).

On a participant level, response was defined as follows: CR refers toregression of all dysplastic lesions found at baseline to no higher thanhyperplasia as defined by the site by site analysis at six months and noappearance of new dysplastic lesions that were mild dysplasia or worse.PD was defined as progression of one or more sites by two or more gradesas defined for the lesion-specific analysis above or appearance of newdysplastic lesions that were mild dysplasia or worse at six months.Partial response (PR) was defined as regression of some but not all ofthe dysplastic lesions but no appearance of new lesions that were milddysplasia or worse. Stable disease (SD) referred to subjects who did nothave a CR, PR or PD.

For quantitative nuclear morphometry, the median MI for histologicallynormal biopsies was 1.25. The inter-observer variation of measuring theMI was such that a change in MI greater than 0.11 (>2SD) was taken to bea significant change. For the lesion specific analysis, CR was definedas regression of a lesion with a MI of >1.36 to ≦1.36. PD was defined asincrease in the MI from ≦1.36 to >1.36. On a participant by participantbasis, CR was defined as regression of all sites with MI>1.36 to ≦1.36.PD was defined as progression of any site similar to that defined underthe site by site analysis or appearance of new lesions with a MI of>1.36.

Descriptive statistics were used to summarize subject characteristics,and pathologic evaluations of the bronchial biopsy examinations.Comparison between treatment arms was done with the Mann-Whitney testfor continuous variables such as age, smoking intensity (pack-years) andMI. Pearson's χ² test with continuity correction was used to comparecategorical variables such as gender, smoking status (current versusformer smokers) and response rates (progression and regression) in thetwo arms. Fisher's exact test was applied to the lower partial responseand stable disease rates. All P values are two-sided. A two-sided Pvalue of less than 0.05 was considered statistically significant.

To adjust for the effect of various pre-treatment factors on thelikelihood of regression or progression of dysplastic lesions, amultiple logistic regression analysis was used on a participant level.This analysis included the following variables: age, sex, smoking statusand the smoking intensity (pack-years). All analyses were unconditional,and tests of statistical significance and confidence intervals (Cis) forodds ratios (ORs) were based on the log-likelihood test.

Results

Summary of the results : In the lesion specific analysis, progression ofpre-existing dysplastic lesions by two or more grades and/or appearanceof new lesion was 9% lower in the ADT group (8% ADT versus 17% placebo,P<0.001). In the person specific analysis, the progression rate was 22%lower (32% ADT versus 59% placebo, P=0.01). Nuclear morphometry of thebiopsies was used as a secondary endpoint. In half of the participantswho had elevated morphometry indices pre-treatment, the progression ratewas 19% lower (41% ADT versus 60% placebo, P=0.28). Adverse events weremostly minor gastrointestinal symptoms that resolved with dose reductionor discontinuation of the medication.

Detailed Results:

Of the 112 subjects that were randomly assigned to ADT or placebo, 61were allocated to the ADT arm and 51 to the placebo arm (FIGURE). Onehundred and two subjects completed the six months study. Ten subjectsdropped out unrelated to side effects of the study medication. They didnot return for the six months follow-up bronchoscopy and hence wereexcluded from the analyses since changes of the histopathology grades ofthe bronchial biopsies were used as the primary endpoint to assess theefficacy of the chemopreventive treatment An additional subject in theplacebo group was excluded from the analysis because the follow-upbiopsy of the same site that showed dysplasia prior to treatment hadincomplete epithelium for making a pathological diagnosis. The remaining101 subjects who had taken one or more doses of the study medication andhad a follow-up bronchoscopy after six months of intervention wereincluded in the analysis.

The characteristics of the 101 participants who completed the trial areshown in Table 1. There was no significant difference in the age, sexand smoking history between the ADT and placebo groups. There were fewerformer smokers in the placebo arm (18% versus 34% in the ADT group) butthe difference was not significant (p=0.07). TABLE 1 Characteristics ofParticipants in this Study ADT Placebo Number of Participants 56 45 Age(Years) Median 54 54 Range 41-74 43-71 Gender Male 36 (64%) 26 (58%)Female 20 (36%) 19 (42%) Smoking Median 48 47 (Pack-Years) Range 30-9932-172 Current 37 (66%) 37 (82%)* Smoker Former Smoker 19 (34%)  8 (18%)Highest Grade Mild 44 (79%) 37 (82%) Dysplasia Moderate/Severe 12 (22%) 8 (18%) Number With ≧ 2 Sites of Dysplasia 37 (66%) 29 (64%)*P = 0.07 compared to Anethole dithiolethione (ADT) group by chi-squaretest.

Effects of Sialor on Histopathology

Lesion Specific Analysis

At six months, the complete response rate was 12% higher in the ADTgroup than the placebo group (53% versus 41%, p=0.14, not significant).The progression rate was significantly lower in the ADT group than theplacebo group (8% versus 17%, p<0.001) (Table 2A). TABLE 2 PathologyGrades of Bronchial Biopsies Before And Six Months After Intervention:Lesion Specific and Person Specific Analyses. A. Lesion SpecificAnalysis 6 Months Follow-up Pathology Grade of Bronchial BiopsiesBronchial Biopsies 1 2 3 4 5 Total Baseline Placebo Not sampled+ 17 12 19 4 0 52 1 82 4 14 0 0 100  2 10 2  8 0 0 20 3 36 13  33 1 1 84 4  3 3 3 1 0 10 5  0 0  0 0 0  0 Total 148  34  77 6 1 266  Baseline ADT Notsampled 34 8 13 0 1 56 1 100  9  5 2 0 116  2 22 2  3 0 0 27 3 53 14  276 0 100  4  8 0  2 3 1 14 5  0 0  2 0 0  2 Total 217  33  52 11  2 315 *1 = normal/hyperplasia, 2 = metaplasia, 3 = mild dysplasia, 4 =moderate dysplasia, 5 = severe dysplasia. ⁺Not sampled refers toadditional biopsies taken at the six months follow-up bronchoscopy. Boldareas on the left represent regression (CR), only dysplasia sites couldregress to normal/hyperplasia. Bold areas on the right representprogression (PD). Any site at baseline could progress. New dysplasticlesions not sampled at baseline were considered as PD as the enrollmentcriteria was bronchial dysplasia. CR was 53% (61/116) and 41% (39/94) inthe ADT and placebo groups respectively, P = 0.14, χ² test). PD was 8%(24/315) and 17% (46/266) in the ADT and placebo groups respectively, P< 0.001, χ² test). All P-values are two-sided. B. Person SpecificAnalysis CR PR SD PD Excluded Total Placebo  7  7  4 26 1⁺ 44 (16%)(16%) (9%) (54%) ADT 15 13 10 18 56 (27%) (23%) (18%) (32%) p-values* 0.29 NA NA  0.013 *Comparison of proportions with chi-square test. AllP values are two-sided. NA: not applicable ⁺One Placebo participantwhose baseline dysplasia sites could not be scored at 6 months becauseof incomplete epithelium was omitted from the comparison.

Person Specific Analysis

The complete response rate was 11% higher in the ADT group versusplacebo, (27% versus 16%, p=0.29). The PD rate was 22% lower for the ADTgroup versus the placebo group (32% versus 54%). The difference wasstatistically significant, P=0.013, χ² test)(Table 2B). Seven of the 37current smokers (19%) in the ADT group and 6/37 current smokers (16%) inthe placebo group gave up smoking during the study. Eleven of the 37current smokers in the ADT group increased their smoking during thestudy and 15 decreased their smoking. The corresponding FIGURES for theplacebo group were 6/37 and 21/37, respectively. The average percentagechange in smoking compared to baseline was 34% in the ADT group and 37%in the placebo group.

Multiple logistic regression analysis was used to determine simultaneouseffect of age, gender, smoking history and effect of treatment onprogression (PD) (Table 3). TABLE 3 Likelihood Of Progressive DiseaseSix Months After Intervention 95% Confidence Significant factors: OR*Interval P-value Treatment Placebo 3.5 1.3-9.3 0.01 ADT 1 Gender Men 6.22.1-17.8 0.001 Women 1 Smoking Current- 3.4 1.1-10.8 0.03 status smokerFormer 1 smoker Cigarettes Smoked 1.0 1.0-1.1 0.07 (per 1 pack-year)*Multiple regression analysis

Placebo subjects were estimated to have 3.5 times higher odds ofprogression (95% Cl=1.3-9.3, p=0.013). Current smokers had 3 time higherodds of PD than ex-smokers (95% Cl=1.1-10.8, p=0.03). Gender wasestimated to have the strongest effect on PD with men having 6 timehigher odds of PD than women (95% Cl=2.1-17.8, p=0.001).

Multiple logistic regression analysis was also used to determinesimultaneous effect of age, gender, smoking history and effect oftreatment on regression (CR). ADT treatment was not significantlyassociated with increasing odds of CR. Smoking as measured by pack-yearsdecreased the odds of CR by 10% for each additional 1 pack-year ofsmoking (OR 0.9, 95% Cl=0.84-0.96, p=0.002). This means that a personwith 10 pack-year heavier smoking was estimated to have 64% lower oddsof CR than another subject with similar age and gender but less smoking.For a person 10 years older at baseline (given the same smokingintensity and gender), the model estimated 3.3 fold higher odds of CR(95% Cl=1.6-7.1, p=0.002). Gender had a borderline significantassociation with CR suggesting that females had on average 3 timeshigher odds of CR (95% Cl 0.96-9.3, p=0.06).

Effect of Dose Reduction on Histopathology

Thirty-one subjects were able to take the full 25 mg TID dose of ADT for6 months. The dose was reduced to 25 mg BID in 17 subjects, 25 mg QD in4 subjects and discontinued in 4 subjects. There was no significantdifference in the CR or PD rates (all P-values >0.90) between those whowere on the full dose compared to those on a reduced dose (Table 4).TABLE 4 Effect of Dose Reduction on Response Rates Dose Reduction CR PRSD PD Total No  9 (29%)  5 (16%)  7 (23%) 10 (32%) 31 Yes  6 (24%)  8(32%)  3 (12%)  8 (32%) 25 Total 15 13 10 18 56 P-value*  0.91 NA  0.98*Chi-square testNA: not applicable

Effect of ADT on Nuclear Morphometry

Fifty-two percent of the subjects in the ADT group and 57% of thesubjects in the placebo group had at least one biopsy that had aMI>1.36. In the person specific analysis, the CR rate was 21% higher inthe ADT group than in the placebo (45% versus 24%, p=0.19). The PD ratewas 19% lower (41% versus 60%, p=0.28)(Table 5). TABLE 5 NuclearMorphometry of Bronchial Biopsies Before and Six Months afterIntervention: Lesion Specific and Person Specific Analyses A. LesionSpecific Analysis Baseline 6 months Biopsies MI ≦1.36 >1.36 totalPlacebo ≦1.36  66 32 (33%)  98 (100%) >1.36  19 (63%) 11  30 (100%)Total  85 43 128 ADT ≦1.36  85 27 (24%) 112 (100%) >1.36  29 (76%)  9 38 (100%) Total 114 36 150 Comparison of regression rates between ADTand Placebo p = 0.37, chi-square test Comparison of progression rates p= 0.22, chi-square test B. Subject Specific Analysis CR PR SD PD TotalPlacebo  6 (24%) 1 (4%) 3 (12%) 15 (60%) 25 (100%) ADT 13 (45%) 1 (3%) 3(10%) 12 (41%) 29 (100%) p-values 0.19* NA 0.28* *Comparison usingchi-square test for equality of proportions NA: not applicable

In the lesion specific analysis, the CR rate was 76% in the ADT groupand 63% in the placebo group. The corresponding PD rates were 24% and33% respectively. The difference between the two groups was notsignificant (P=0.37 and 0.22 for CR and PD, respectively). (Table 5).

Adverse Events

Symptoms of excessive flatus, abdominal bloating, loose stool andconstipation were frequently reported by participants in both groups(Table 6). TABLE 6 Adverse Events Symptom Placebo ADT Excessive Flatus53%  93% Abdominal Bloating 4%  25%* Loose Stool 8% 16% Diarrhea 12% 13% Constipation 0%  5% Increased Saliva 4%  2%*P = 0.01 compared to placebo (chi-square test)

Only abdominal bloating was significantly more frequent in the ADT group(P=0.018). Grade 2 symptoms were observed in 51% of the participantstaking ADT versus 20% of those on placebo. Grade 3 symptoms wereobserved in 11% of those taking ADT and 6% of those on placebo. One ofthe participants taking ADT had a Grade 3 elevation of liver enzymes.The liver enzymes returned to normal after discontinuation of the ADT.Dose reduction was required in 45% of the participants taking ADT and25% of those on placebo. The study medication had to be discontinued infour of the subjects in the ADT group and one of the subjects in theplacebo group because of complaints of minor, though intolerablegastrointestinal symptoms.

Discussion

In the primary endpoint analyses, a significantly lower rate ofprogression of pre-existing dysplastic lesions by two or more gradesand/or appearance of new lesions were observed after 6 months of ADT ata dose of 25 mg orally TID compared to the placebo.

At the present time, bronchial dysplasia is one of the best surrogateendpoint biomarker to assess the effect of new chemopreventive agents.The morphological criteria for pre-invasive lesions have been defined inthe recent WHO classification (Travis et al., 1999). Grading of squamouspre-invasive lesions was found to be reproducible. In humans, thepresence of dysplastic cells in sputum cytology or bronchial biopsy isassociated with the development of invasive lung cancer in prospectivestudies (Frost et al., 1986; Saccomano et al., 1982; Risse et al., 1988;Melamed et al., 1982; Thiberville et al., 1997; Shibuya et al., 2001)similar to what is known from cancer progression models in animals(Nasiell et al., 1987). Reversal of dysplasia with successful modulationis associated with reduced cancer risk (Boone et al., 1992; Boone etal., 1997). To minimize inter-observer variation, dysplastic changes inthe bronchial epithelium can be quantitated by image cytometry (Lam etal., 1998; Boone et al., 1992; Boone et al., 1997) as is done in thecurrent study. Using quantitative nuclear morphometry we observed a 21%better CR rate and a 19% lower PD rate in the ADT group versus placebo.Although the differences were not statistically significant, themagnitude of the difference between the two groups was similar to thatobserved using histopathology criteria (Travis et al., 1999). A greaterstatistical significance might have been observed if both histopathologyand nuclear morphometry were used as the enrollment criteria as only 50%of our subjects had at least one biopsy with MI>1.36 at baseline. Theinventors did not use nuclear morphometry in the inclusion criteria inthis clinical trial because it was developed after the study began.

This is the first Phase II lung cancer chemoprevention trial usingbronchial dysplasia as the primary intermediate endpoint biomarker.Previous studies used the metaplasia index or a combination ofmetaplasia and dysplasia with very few dysplastic lesions present in theparticipants (Lee et al., 1994; Kurie et al., 2000). Using metaplasia asthe intermediate endpoint biomarker, isotretinoin andN-(4-Hydroxyphenyl)retinamide were not found to be effectivechemopreventive agents (Lee et al., 1994; Kurie et al., 2000). Theeffect of these agents on bronchial dysplasia is not known. Similar tothese studies of retinoids (Lee et al., 1994; Lippman et al., 2001), inaddition to ADT effect, the inventors observed a difference betweenformer and current smokers in the development and progression ofdysplastic lesions. The reason why women had better response than men isnot known and requires further investigation.

The mechanisms of action of ADT are multiple. It exerts effects onglutathione. After administration of ADT, an increase of intracellularglutathione has been shown in animals pretreated with various toxins aswell as in normal animals. This effect is produced by a stimulation ofglutathione synthesis via the glutamyl cysteine synthetase and isaccompanied by an increase of glutathione-dependent enzyme activity(Warnet et al., 1989). Increase of glutathione-S-transferase activity, aphase II enzyme implicated in chemoprevention against aflatoxinhepatocarcinogenesis, induced by ADT, can explain its protective actionagainst aflatoxin tumorigenicity observed in rats by Kensler et al.,1987. It is also a potent inhibitor of lipid peroxidation, asdemonstrated in-vitro in a rat liver microsome model. It exerts freeradical scavenger properties evidenced among others by radiolysisstudies performed in various conditions (Christen et al., 1995; Christenet al., 1996), and thereby it protects the cellular membrane byinhibiting lipid peroxidation and diene formation. Moreover, ADT wasobserved to be capable of modulating the nuclear factor NFκB (a redoxsensitive cytolytic transcription factor) activation in human Jurkatcells (Sen et al., 1996). Pretreatment of human Jurkat cells by ADTsignificantly protects cells against oxidative stress-inducedcytotoxicity (Khanna et al., 1998). Although dithiolethiones may act viaseveral mechanisms including inhibition of cell replication, it appearsthat they act predominantly by carcinogen detoxification viaup-regulation of phase II enzymes including glutathione-S-transferase(GST). In a chemoprevention setting using a rat model of hepatoma, themean GST levels in the liver were statistically increased (p<0.01) bothby ADT and Oltipraz. The GST level was found to be elevated by 3.2 to4.5-fold after changing the concentration of Oltipraz in the diet from0.01% to 0.1% (Kensler et al., 1987). In the human, a study in Qidong,China showed that intermittent, high-dose Oltipraz inhibited phase Iactivation of aflatoxins, and sustained low-dose Oltipraz increasedphase II conjugation of aflatoxin (Wang et al., 1999). Thedetoxification and anti-oxidant actions may explain the greater effectof ADT in preventing appearance of new dysplastic lesions or progressionof existing dysplastic lesions to a higher grade than regression ofexisting dysplastic lesions observed in this study. Administration ofADT longer than 6 months in a larger number of participants may show theeffect of ADT on regression of existing dysplastic lesions better.

In this Phase II study, the inventors used the dosage of ADT that wasapproved by Health Canada for the treatment of xerostomia in order toestablish the potential efficacy of this agent in smokers with bronchialdysplasia. The only adverse events observed were gastrointestinal. Thisis in keeping with previous clinical data on the safety profile of ADTin the treatment of patients with dry mouth (Remick et al., 1983). Sincethe activity of the drug was observed despite 45% of the participantswere taking the medication only twice or once a day, the possibilitythat effective chemopreventive activity can be achieved in a two times aday or once daily dosage needs to be investigated further.

This study showed for the first time in smokers with pre-malignantlesions that ADT (Sialor®, Sulfarlem®) statistically reduces theappearances of new dysplastic lesion or progression of pre-existingdysplastic lesions. Given that more than half of all long-term smokersare unable to stop smoking despite aggressive behavioral andpharmacologic smoking cessation measures (Wang et al., 1999), this studysuggests new strategies for lung cancer control using chemoprevention bythe free radical scavenger and glutathione inducer anetholedithiolethione (Sialor®, Sulfarlem®).

REFERENCES

-   Bolton M G, Munoz A, Jacobson L P, Groopman J D, Maxuitenko Y Y,    Roebuck B D, et al. Transient intervention with Oltipraz protects    against aflatoxin-induced hepatic tumorigenesis. Cancer Res 1993;    53:3499-504.-   Boone C W, Bacus J W, Bacus J V, Steele V E, Kelloff G J. Properties    of intraepithelial neoplasia relevant to the development of cancer    chemopreventive agents. J Cell Biochem, Suppl 1997;28/29:1-20.-   Boone C W, Kelloff G J, Steele V E. Natural history of    intraepithelial neoplasia in humans with implications for cancer    chemoprevention strategy. Cancer Res 1992;52:1651-1659.-   Christen M O. Anethole Dithiolethione: Research overview and    perspectives. In: Packer L, Traber M G, Xin W, editors. Molecular    Mechanisms and Health Effects. AOCS Press; 1996. p. 236-242.-   Christen M O. Anethole dithiolethione: Biochemical considerations.    Methods in Enzymology 1995;252:316-323.-   Doudkine A, MacAulay C, Poulin N, Palcic B. Nuclear texture    measurements in image cytometry. Pathologica 1995;87(3):286-289.-   Epstein J B, Decoteau W E, Wilkinson A. Effect of Sialor in    treatment of xerostomia in Sjogren's syndrome. Oral Surg Oral Med    Oral Pathol 1983;56:495-9.-   Frost J K, Ball W C Jr, Levin M I, Tockman M S, Erozan Y S, Gupta P    K, et al. Sputum cytology: use and potential in monitoring the    workplace environment by screening for biological effects of    exposure. J Occup Med 1986;28:692-703.-   Garner D M, Harrison A, MacAulay C, Palcic B. Cyto-Savant™ and its    use in automated screening of cervical smears. In: Wied G L, Bartels    P H, Rosenthal P H, Schenck U, editors. Compendium on the    Computerized Cytology and Histology Laboratory. Chicago: Tutorials    of Cytology; 1994. p.346-352.-   Greenlee R T, Hill-Harmon M B, Murray T, Thun M. Cancer    Statistics, 2001. CA Cancer J Clin 2001;51:15-36.-   Halpern M T, Gillespie B W, Warner K E. Patterns of absolute risk of    lung cancer mortality in former smokers. J Natl Cancer Inst    1993;85:457-464.-   Hong W K, Sporn M B. Recent advances in chemoprevention of cancer.    Science 1997;278:1073-1077.-   Jorenby D E, Leischow S, Nides M, Rennard S, et al. A controlled    trial of sustained-release bupropion, a nicotine patch or both for    smoking cessation. N Engl J Med 1999;340:681-91.-   Kensler T W, Egner P A, Dolan P M, Groopman J D, Roebuck B D.    Mechanism of protection against aflatoxin tumorigenicity in rats fed    5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) and related    1,2-dithiol-3-thiones and 1,2-dithiol-3-ones. Cancer Res    1987;47:4271-7.-   Kensler T W, Groopman J D, Roebuck B D. Chemoprevention by Oltipraz    and other ditholethiones. In: Wattenberg L, Lipkin M, Boone C,    Kelloff G, editors. Cancer Chemoprevention. Boca Raton: CRC    Press; 1992. p. 205-225-   Khanna S, Sen C K, Roy S, Christen M O, Packer L. Protective effects    of anethol dithiolthione against oxidative stress induced    cytotoxicity in human Jurkat T cells. Biochem Pharmacol,    1998;56:61-69.-   Kurie J M, Lee J S, Khuri F R, et al. N-(4-Hydroxyphenyl)retinamide    in the chemoprevention of squamous metaplasia and dysplasia of the    bronchial epithelium. Clin Cancer Res, 2000;6:2973-9.-   Lam S, Kennedy T, Unger M, Miller Y, Gelmont D, Rusch V, et al.    Localization of bronchial intraepithelial neoplastic lesions by    fluorescence bronchoscopy. Chest 1998;113:696-702.-   Lam S, leRiche J C, Zheng Y, Coldman A, MacAulay C, Hawk E, et al.    Sex-related differences in bronchial epithelial changes associated    with tobacco smoking. J Natl Cancer Inst 1999;91:691-696.-   Lam S, MacAulay C E. Endosoopic localization of preneoplastic lung    lesions. In: Martinet Y, Hirsch F R, Martinet N, Vignaud J M,    Mulshine J L, editors. Clinical and Biological Basis of Lung Cancer    Prevention. Switzerland: Birkhauser Verlag Basel; 1998. p.231-237.-   Lee J S, Lippman S M, Benner S E, Lee J J, Ro J Y, Lukeman J M, et    al. Randomized placebo-controlled trial of isotretinoin in    chemoprevention of bronchial squamous metaplasia. J Clin Oncol,    1994;12:937-945.-   Lippman S M, Lee J J, Karp D D, Vokes E E, Benner S E, Goodman G E,    et al. Randomized phase III intergroup trial of isotretinon to    prevent second primary tumors in stage I non-small cell lung cancer.    J Natl Cancer Inst 2001 ;93:605-18.-   Mac Aulay C E, Lam S, Klein-Parker H, Gadzar A, Guillaud M, Payne P,    le Riche J, Dawe C, Band P, and Palcic B. Intermediate endpoint    biomarkers for lung cancer chemoprevention. “PROC.SPIE—Int. Soc.    Opt. Eng. 1998; 3260: 207-210.-   Melamed M R, Zaman M B. Pathogenesis of epidermoid carcinoma of    lung. In: Shimosato Y, Melamed M R, Nettesheim P, editors.    Morphogenesis of lung cancer, Vol I. Boca Raton: CRC Press, 1982. p    37-64.-   Nasiell M, Auer G, Kato H. Cytological studies in man and animals on    development of bronchogenic carcinoma. In: McDowell E M, editor.    Lung Carcinomas. Edinburgh: Churchill Livingstone; 1987. p. 207-242.-   Pendyala L, Schwartz G, Bolanowska-Higdon W, Hitt S, Zdanowicz J,    Murphy M, et al. Phase I/pharmacodynamic study of    N-acetylcysteine/Oltipraz in smokers: early termination due to    excessive toxicity. Cancer Epidemiol Biomarkers Prev 2001;10:269-72.-   Pepin P, Bouchard L, Nicole P, Castoguay A. Effects of sulindac and    Oltipraz on the tumorigenicity of    4-(methynitrosamino)1-(3-pyridyl)-1-butanone in A/J mouse lung.    Carcinogenesis 1992;13:341-348.-   Reddy B S, Rao C V, Rivenson A, Kelloff G. Chemoprevention of colon    carcinogenesis by organosulfur compounds. Cancer Res 1993;53:3493-8.-   Remick R A, Blasberg B, Patterson B D, Carmichael R P, Miles J E.    Clinical aspects of xerostomia. J Clin Psychiatry 1983;44:63-5.-   Risse E K J, Vooijs G P, van't Hof M A. Diagnostic significance of    “severe dysplasia” in sputum cytology. Acta Cytologica    1988;32:629-634.-   Saccomanno G. Carcinoma in situ of the lung: Its development,    detection, and treatment. Semin Respir Med 1982;4(2):156-60.-   Sen C K, Traber K E, Packer L. Inhibition of NF-κB Activation in    Human T-Cell Lines by Anetholdithiolthione. Biochem Biophys Res    Commun 1996;218:148-153.-   Shibuya K, Fujisawa T, Hoshino H: A follow up study of squamous    dysplasia. J Clin Exp Med 2001;199:593-596.-   Sporn M B, Dunlop N M, Newton D L, Smith J M. Prevention of chemical    carcinogenesis by Vitamin A and its synthetic analogs. Fed Proc    1976;35:1332-1338.-   Thiberville L, Metayer J, Raspaud C, Nouvet G. A prospective, short    term follow-up study of 59 severe dysplasias and carcinoma in-situ    of the bronchus using autofluorescence endoscopy. Eur Respir J    1997;10:425S.-   Tong L, Spitz M R, Fueger J J, Amos C A. Lung cancer in former    smokers. Cancer 1996; 78:1004-1010.-   Travis W D, Colby T V, Corrin B, Shimosato Y Brambilla E. Histologic    and graphical text slides for the histological typing of lung and    pleural tumors. In: World Health Organization Pathology Panel: World    Health Organization. International Histological Classification of    Tumors. 3^(rd) ed. Berlin: Springer Verlag; 1999. p 5.-   Wang J S, Shen X, He X, Zhu Y R, Zhang B C, Wang J B, et al.    Protective alterations in phase 1 and 2 metabolism of aflatoxin B1    by Oltipraz in residents of Qidong, People's Republic of China. J    Natl Cancer Inst 1999;91(4):347-54.-   Warnet J M, Christen M O, Thevenin M, Biard D, Jacqueson A, Claude    J R. Protective effect of anethol dithiolthione against    acetaminophen hepatotoxicity in mice. Pharmacol Toxicol    1989;65:63-64.

1. Use of 5-(p-methoxyphenyl)-1,2-dithiole-3-thione or of apharmaceutical derivative thereof for the preparation of a medicamentfor preventing lung cancer in a mammalian subject.
 2. The use of claim1, wherein the subject has precursors of lung cancer.
 3. The use ofclaim 2, wherein the subject has bronchial dysplasia, metaplasia, orpremalignant lesions in the bronchial tree.
 4. The use of any of claims1 to 3, wherein the subject has no history of lung cancer.
 5. The use ofany of claims 1 to 4, wherein the medicament is intended for preventingor reducing the appearance of new dysplastic lesions, or the progressionof pre-existing dysplastic lesions in the subject.
 6. The use of any ofclaims 1 to 4, wherein the medicament is intended for improvingregression of existing dysplastic lesions.
 7. The use of any of claims 1to 6, wherein the pharmaceutical derivative is a pharmaceuticallyacceptable salt of 5-(p-methoxyphenyl)-1,2-dithiole-3-thione.
 8. The useof any of claims 1 to 6, wherein the pharmaceutical derivative is apharmaceutically acceptable metabolite of5-(p-methoxyphenyl)-1,2-dithiole-3-thione.
 9. A method for preventinglung carcinogenesis comprising administering to a mammalian subjecthaving precursors of lung cancer and preferably not having lung cancer(i.e. having no history of lung cancer), a pharmaceutical compositioncomprising a therapeutically effective amount of5-(p-methoxyphenyl)-1,2-dithiole-3-thione or a pharmaceutical derivativethereof.